Lecture 6 Flashcards
What is PCR?
A lab technique to amplify millions/billions of copies of a specific DNA segment using a thermal cycler.
Name three applications of PCR.
DNA sequencing, disease diagnosis (e.g., pathogens), and species identification (e.g., marine organisms).
What are the three main steps of PCR?
Denaturation, Annealing, Extension (repeat for 20–40 cycles).
What happens during denaturation?
DNA strands separate at 94–98°C, breaking hydrogen bonds to create single-stranded DNA.
Describe annealing in PCR.
Primers bind to complementary sequences at 50–65°C. Critical for specificity!
What occurs during extension?
Taq polymerase adds dNTPs at 72°C, synthesizing new DNA strands from primers.
Why are PCR steps repeated cyclically?
To exponentially amplify DNA (doubling copies each cycle).
What are primers?
Short synthetic DNA sequences (18–30 nt) that bind to the target DNA region.
What is the ideal primer length?
18–30 nucleotides for balance between specificity and binding efficiency.
How is Tm calculated?
Tm=2(A+T)+4(C+G). Aim for 60–65°C for forward/reverse primers.
What is optimal GC content for primers?
40–60% to ensure stable binding (avoid too many G/C or A/T).
Why should primers end with G/C?
Stronger binding due to triple hydrogen bonds (vs. A/T’s two).
How to avoid primer-dimers?
Design primers without self-complementary regions or inter-primer homology.
Why is primer design critical?
Poor design causes non-specific amplification or failed reactions.
Design reverse primer for 5’-ATGCCGTA…-3’.
Reverse primer: 3’-…TACGGCAT-5’
How is PCR used in marine research?
DNA barcoding, studying coral symbionts (e.g., zooxanthellae), and tracking microbial diversity.
What is DNA barcoding?
Amplifying specific genes (e.g., COI) to identify marine species.
How does PCR study ocean microbes?
Amplifies 16S rRNA genes to analyze bacterial diversity.
How does PCR study coral symbionts?
Amplifies zooxanthellae DNA to track genetic shifts under stress.
Why is Taq polymerase used in PCR?
Thermostable enzyme from Thermus aquaticus; survives high temps during denaturation.
What is a thermal cycler?
Machine automating PCR’s temperature changes for denaturation, annealing, extension.
How does PCR differ from in vivo replication?
PCR uses heat to denature DNA and synthetic primers; no helicase or RNA primers.
List ideal primer properties.
18–30 nt, Tm 60–65°C, GC 40–60%, G/C at 3’ end, no dimers/hairpins.
How does PCR monitor zooxanthellae?
Amplifies algal DNA to identify stress-tolerant strains in corals over time.
what are primer dimers
when two primers (forward and reverse) anneal to each other instead of binding to the target DNA template
What are Intra-Primer Dimers?
These occur when a single primer molecule folds back on itself and anneals to its own sequence, forming a hairpin-like structure.
What are Inter-Primer Dimers?
These occur when two primer molecules (forward and reverse) anneal to each other instead of binding to the target DNA.
